1. Field of the Invention
There is a continuing need for rapid sensitive methods for determining minute amounts of organic compounds. A number of techniques have been developed toward this end. Among the commercially available techniques are radioimmunoassay, spin-labeled immunoassay, for which reagents are sold under the trademark FRAT.RTM., homogeneous enzyme immunoassay, for which reagents are sold under the trademark EMIT.RTM., and hemagglutination (HI). These techniques are effective for determining amounts of materials in the range of 10.sup.-6 to 10.sup.-10 M or less.
These techniques all involve the ability of a receptor molecule, usually an antibody, to be able to recognize a specific spatial and polar organization of a molecule. Except for hemagglutination, the techniques depend upon providing a reagent which can compete with the molecule being assayed for the receptor. By being able to distinguish between the reagent which is bound to receptor and reagent which is unbound, one can determine the amount of the compound of interest which is present.
In developing immunoassays, one is limited by the availability and properties of an appropriate receptor. However, as for the other reagents and the technique of measurement, there are a number of different considerations which make for a more accurate, convenient or commercially desirable assay. First, it is desirable that there be a minimum number of measurements of the various reagents, as well as transfers of the various reagents. Secondly, the equipment for measuring should be reasonably economical, so as to be accessible to a broad range of users. Thirdly, the reagents employed should be relatively stable, so as to be capable of storage and shipment. Fourthly, the method should not be subject to significant interference from other materials which may be adventiously present in the sample to be assayed. Other considerations are ease of training of technicans, absence of health hazards, sensitivity, reproducibility, and applicability to a wide variety of ligands.
The subject invention is predicated on the phenomenon of energy transfer between two chromophores. When a fluorescing chromophor is irradiated with light absorbed by the chromophore, the fluorescing chromophore can dissipate the energy of the absorbed light by emitting light of longer wavelength, that is, fluorescing. If another chromophore is within less than 100A of the fluorescer and absorbs light at the wavelength of emission, there is a probability, depending upon other factors, that the fluorescer will transfer to the other chromophore the energy which would otherwise have been emitted as light, in effect, quenching the fluoroescer.
2. Description of the Prior Art
U.S. Pat. No. 3,709,868 is exemplary of a radioimmunoassay. U.S. Pat. No. 3,690,834 is exemplary of a spin immunoassay. U.S. Pat. Nos. 3,654,090 and 3,817,837 are exemplary of enzyme immunoassays. Articles of interest include an article by Ludwig Brand and James R. Gohlke, entitled, Fluorescence Probes for Structure, Annual Review of Biochemistry, 41, 843-868 (1972); and Stryer, Science, 162, 526 (1968). Also of interest is co-pending application Ser. No. 402,693, filed Oct. 2, 1973.